Members of the fungal genus Pneumocystis can cause a lethal pneumonia (PCP) in hosts with debilitated immune systems, such as HIV-infected persons with AIDS;patients undergoing immunosuppressive therapy;and more recently in those patients received targeted immunotherapy. The manner in which the infection is disseminated;the life cycle within the mammalian lung;and the strategies used for survival within this inhospitable environment are largely unknown due in large part to the lack of a continuous cultivation method. It is our long term goal to understand the infection process of Pneumocystis as a means to identify its survival strategies which could then be exploited for interdiction of infection. Production of biofilms is a strategy used by many microbes for protection against environmental assaults;for communication and differentiation among members;and as foci for dissemination. We posit that the attachment and growth of Pneumocystis within the lung alveoli is akin to biofilm formation. An in vitro system was identified that supports apparent biofilm formation by P. carinii (from rat) and P. murina (from mouse). The biofilms showed similar growth kinetics, confocal attributes, and morphological changes compatible with biofilm formation by other fungi. In the present proposal, this system will be optimized to provide a novel tool for understanding the survival strategies of Pneumocystis and then will be used to address questions about the biology of the process. The following specific aims are proposed: (1) Define and characterize the optimal conditions for formation of Pneumocystis biofilms. A systematic approach will be used to evaluate matrices, conditions, and additives leading to robust biofilm formation. Microscopic and quantitative methods will be used to chart the progression of biofilm formation. (2) Identify the biological process associated with biofilm formation. The morphologic changes Pneumocystis undergoes as it forms a biofilm are dramatic. Identification and verification of the genes and gene products involved in the progression using molecular and biochemical methods will provide basic information on the biology of biofilm production and lead to a new appreciation and understanding of this process. (3) Assess the pathogenicity of in vitro biofilms in vivo. The progression of infection initiated by biofilm-derived organisms vs. the standard model will be compared by microscopic and quantitative methods. The in vivo morphology and gene expression will compared to in vitro biofilms for validation of the in vitro system. Optimization and evaluation of the biofilm process will fundamentally advance the study of Pneumocystis. PUBLIC HEALTH RELEVANCE: Microbial pathogens use biofilms to escape the infected host's immune defenses and antimicrobial therapy. We identified a biofilm system for the fungal pathogen, Pneumocystis, that will be used to understand the strategies it employs to survive in the lung and cause pneumonia.